Immunoelectron microscopic localization of the electrogenic Na/HCO2 cotransporter in rat and ambystoma kidney
Immunoelectron microscopic localization of the electrogenic Na/HCO2 cotransporter in rat and ambystoma kidney
复制标题
DOI：
10.1681/asn.v11122179
复制
发表时间：
2000-12-01
影响因子：
13.6
通讯作者：
Aalkjær, C
中科院分区：
文献类型：
--
作者：
Maunsbach, AB;Vorum, H;Aalkjær, C
免疫荧光分析表明，产电Na+/HCO 3-（NBC 1）在大鼠肾脏的近端小管和蝾螈Ambystoma tigrinum肾脏的近端和远端小管中表达。本研究采用高分辨率免疫电镜技术对NBC 1在大鼠和钝口吸虫肾脏的亚细胞定位进行了详细的研究。为此，开发了针对大鼠肾脏C末端（rkNBC 1）氨基酸928至1035和氨基酸1021至1035的两种兔多克隆抗体。亲和纯化的抗体揭示了一个强大的带约140 kD的免疫印迹膜从大鼠肾皮质，但没有信号分离的膜从外部和内部髓质。去糖基化将表观分子量降低至约120 kD，与预测分子量相对应。从钝口螈肾的外侧部分分离的膜中也存在类似但较弱的条带。在大鼠肾脏中，免疫组织化学证实了rkNBC 1在近端小管的曲节中的存在。在大鼠肾皮质的冷冻切片或Lowicryl HM 20切片中，不同的免疫金标记与近端小管S1和S2段的基底外侧质膜相关，而在S3中未观察到标记。基底和侧质膜的标记密度相似，并且与膜的内表面特异性相关，与转运蛋白C-末端的内部位置一致。与此相反，rkNBC 1是不存在的顶端质膜，并没有观察到在细胞内囊泡，包括那些密切相关的基底侧质膜。在Ambystoma肾，一个弱的标记存在于近端小管的基底外侧膜和较强的标记，观察到在晚期远段。结果表明，rkNBC 1仅在大鼠近曲小管的S1段和S2段以及钝口鱼近曲小管和远曲小管中表达，rkNBC 1存在于基底和侧质膜中，而在顶端质膜的胞内囊泡中不表达。
Immunofluorescence analysis has revealed that electrogenic Na+/HCO3- (NBC1) is expressed in the proximal tubule of rat kidney and in the proximal and distal tubules of the salamander Ambystoma tigrinum kidney. The present study was undertaken to define the detailed subcellular localization of the NBC1 in rat and Ambystoma kidney using high-resolution immunoelectron microscopy. For this purpose, two rabbit polyclonal antibodies raised against amino acids 928 to 1035 and amino acids 1021 to 1035 of the C-terminus of rat kidney (rkNBC1) were developed. The affinity-purified antibodies revealed a strong band of approximately 140 kD in immunoblots of membranes from rat kidney cortex but no signal in membranes isolated from outer and inner medulla. Deglycosylation reduced the apparent molecular weight to approximately 120 kD, corresponding to the predicted molecular weight. A similar but weaker band was also present in membranes isolated from the lateral part of Ambystoma kidney. In rat kidney, immunohistochemistry confirmed the presence of rkNBC1 in convoluted segments of the proximal tubules. In ultrathin cryosections or Lowicryl HM20 sections from rat kidney cortex, distinct immunogold labeling was associated with the basolateral plasma membrane of segments S1 and S2 of proximal tubules, whereas in S3 no labeling was observed. The labeling density was similar at the basal and lateral plasma membrane and was specifically associated with the inner surface of the membrane consistent with the internal position of the C-terminus of the transporter. In contrast, rkNBC1 was absent from the apical plasma membrane and not observed in intracellular vesicles, including those closely associated with basolateral plasma membrane. In Ambystoma kidney, a weak labeling was present in the basolateral membrane of the proximal tubule and stronger labeling was observed in the late distal segment. The results demonstrate that rkNBC1 is expressed only in segment S1 and segment S2 of rat proximal tubule as well as Ambystoma proximal and late distal tubule and that rkNBC1 is present in both basal and lateral plasma membranes and absent in intracellular vesicles of the apical plasma membrane.